PsbD | D2 protein of PSII
AS06 146 | Clonality: Polyclonal | Host: Rabbit | Reactivity: [global antibody] for A. thaliana, Anabaena 7120, D. brightwellii, H. vulgare, C.reinhardtii, C. zofingiensis, L. corniculatus, N. tabacum, O. sativa, P. sativum, P. vulgaris, P. tricornutum, T. pratense, S. alba, Synechococcus sp. PCC 7942, Synechocystis sp. PCC6803, T. guillardii, T. pseudonana, Triticale, U. prolifera, Z. mays
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Product Information
KLH-comjugated synthetic peptide derived from the C-terminal of known PsbD sequences including Arabidopsis thaliana P56761, Hordeum vulgare P11849, Chlamydomonas reinhardtii P06007, Synechococcus sp. PCC 7002 P20898
Reactivity
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Application examples
Application example
2 µg of total protein from (1) Arabidopsis thaliana leaf extracted with Agrisera Protein Extraction Buffer, PEB (AS08 300), (2) Hordeum vulgare leaf extracted with PEB, (3) Chlamydomonas reinhardtii total cell extracted with PEB, (4) Synechococcus sp. 7942 total cell extracted with PEB, (5) Anabaena sp. total cell extracted with PEB were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% blocking reagent in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 50 000 for 1 h/RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS09 602) diluted to 1:50 000 in 2% blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with chemiluminescent detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).
1 μg of chlorophyll from Pisum sativum (1), Zea mays, mesophyll (2) and bundle sheath (3), Echinochloa crus-galli, mesophyll (4) and bundle sheath (5), Arabidopsis thaliana (6) chloroplasts extracted with 0.4 M sorbitol, 50 mM Hepes NaOH, pH 7.8, 10 mM NaCl, 5 mM MgCl 2 and 2 mM EDTA were loaded to lanes. Samples were denatured with Laemmli buffer at 75°C for 5 min and were separated on 12% SDS-PAGE, and blotted 30 min to PVDF using wet transfer. Blot was blocked with 5% milk for 2h at room temperature (RT) with agitation. Blot was incubated in the primary antibody Anti-PsbD at a dilution of 1: 5000 in 1% milk in TBS-T overnight at 4°C with agitation. The antibody solution was decanted and the blot was washed 4 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG HRP conjugated, from Agrisera, AS09 602) diluted to 1:20 000 in 1 % milk in TBS-T for 1h at RT with agitation. The blot was washed 5 times for 5 min in TBS-T and 2 times for 5 min in TBS, and developed for 1 min with 1.25 mM luminol, 0.198 mM coumaric acid and 0.009% H 2 O 2 in 0.1 M Tris- HCl, pH 8.5. Exposure time in ChemiDoc System was 14 seconds.
Courtesy of Dr. Wioleta Wasilewska-Dębowska, University of Warsaw, Poland
Quantity control of chlorophyll amount loading onto gel. From 0,25 μg to 2,0 μg of chlorophyll from Pisum sativum chloroplasts were loaded to lanes. All steps of experiments were the same as described above.
Courtesy of Dr. Wioleta Wasilewska-Dębowska, University of Warsaw, Poland

Reactant: Plant
Application: Western Blotting
Pudmed ID: 27590049
Journal: BMC Plant Biol
Figure Number: 9A
Published Date: 2016-09-02
First Author: Mazur, R., Sadowska, M., et al.
Impact Factor: 4.142
Open PublicationChanges of PSII and PSI antenna and core protein levels. Proteins from control and Tl-treated white mustard leaves were separated by SDS-PAGE followed by immunodetection with antibodies against Lhcb1, Lhcb2, Lhca1 (antenna proteins) and D1, D2, CP43, PsbO, PsaA (core proteins). Samples were loaded on the equal amount of chlorophyll (0.25 ?g). Description of samples abbreviation as given in the legend to Fig. 3

Reactant: Chlamydomonas reinhardtii (Green Alga)
Application: Western Blotting
Pudmed ID: 28466860
Journal: Nat Commun
Figure Number: 5A
Published Date: 2017-05-03
First Author: Fu, H. Y., Picot, D., et al.
Impact Factor: 13.783
Open PublicationAccumulation of photosynthetic complexes in the mutant strains.Cells were grown in TAP medium at 25?°C under LED white light (8??mol photons m?2?s?1) and collected at the mid-log phase. Two independent lines are shown for each construct. Protein samples were loaded on an equal chlorophyll basis (0.5??g per lane), and a dilution series of WT samples is shown for semi-quantitative comparison. Antibodies against essential subunits of PSII (D2), PSI (PsaA), cytochrome b6f (PetA) and ATP synthase (AtpB) probed the accumulation of the respective photosynthetic complexes. Numbers on the left side of the blots are molecular weights in kD. See Supplementary Fig. 9 for the uncropped blot images.

Reactant: Arabidopsis thaliana (Thale cress)
Application: Western Blotting
Pudmed ID: 31240258
Journal: Commun Biol
Figure Number: 2A
Published Date: 2019-06-27
First Author: Pralon, T., Shanmugabalaji, V., et al.
Impact Factor: None
Open PublicationThylakoid protein phosphorylation and state transitions are disturbed after high light treatment in pgr6 background. a Total protein extracts of 4-week-old wild type (WT), pgr6-1, pgr6?2, sps2 and stn7/stn8 analysed by immunoblotting with anti-phosphothreonine antibody; the principal thylakoid phospho proteins are indicated on the right according to their size. Core photosystem II proteins D1 (PsbA) and D2 (PsbD) are indicated as a single band due to their poor resolution. Actin was used as a loading control. b Lhcb1 and Lhcb2 phosphorylation levels were visualised after separation on Phostag™-pendant acrylamide gels. The upper band corresponds to the phosphorylated form (p-), stn7/stn8 double mutant is a non-phosphorylated control. c Average transient of the variable room temperature chlorophyll fluorescence measured during the transition from red (660nm) supplemented with far-red light (720nm) state 1 to pure red light state 2 (n?=?4 independent pots containing 2–3 plants). The fluorescence curves from pgr6 and sps2 are shifted on the x-axis to allow visualising the FMST1 and FMST2 values. The x-axis time scale refers to the wild-type curve. d Calculated quenching related to state transition (qT?=?(FMST1?–?FMST2)/FM), expressed as the percentage of FM that is dissipated by the state 1 to state 2 transition, of wild type (WT), pgr6?1 and sps2 under moderate light (120?mol?m?2?s?1) (ML) and after 3?h of high light (500?mol?m?2?s?1) (HL). Whiskers and box plot shows the minimum, first quartile, median, average, third quartile and maximum of each dataset (n?=?4 biologically independent samples); p-values are calculated via a two-tailed Student’s t test. e STN7 phosphorylation level visualised after separation on Phostag™-pendant acrylamide gels. The upper band corresponds to the phosphorylated form (p-), a protein sample from stn7/stn8 double mutant was loaded as a control for the antibody specificity. Uncropped images of the membranes displayed in a, b and e are available as Supplementary Fig. 11. Data points for items c, d are available as Supplementary data 2

Reactant: Arabidopsis thaliana (Thale cress)
Application: Western Blotting
Pudmed ID: 32963291
Journal: Sci Rep
Figure Number: 3C
Published Date: 2020-09-22
First Author: Wang, L., Leister, D., et al.
Impact Factor: 4.13
Open PublicationPhotomorphogenesis is not significantly affected in ros1, nrpd1, nrpe1, rdr2 or ago4 mutant seedlings. (a) Phenotypes (upper panel) and the corresponding maximum quantum yields of PSII (Fv/Fm) (lower panel) of 4-day-old etiolated seedlings. Fv/Fm was measured with an imaging Chl fluorometer (Imaging PAM). Scale bar?=?1 cm. (b) Phenotypes (upper panel) and corresponding Fv/Fm values (lower panel) of 3-day-old etiolated seedlings which had been exposed to continuous light for 24 h. (c) Immunoblot analysis of the PSII core proteins (D1 and D2), Lhcb1 and FLU during greening of etiolated seedlings. WT seedlings were grown for 3 days in the dark and exposed to light for between 0 and 48 h, as indicated. Extracted total proteins were normalized with respect to fresh weight and fractionated by SDS-PAGE. Blots were then probed with antibodies raised against the individual proteins. Total proteins from 5-day-old WT seedlings grown under continuous light (LL) and LD conditions (LD) were used as positive controls. The total protein accumulation of each sample was visualized by staining the gel with Coomassie Blue R250 (C.B.). The figure was assembled from different blots (delineated by a black rectangle) and full-length blots are presented in Supplementary Fig. S7. (d) Immunoblot analysis of representative photosynthesis proteins of 3-day-old etiolated mutant seedlings which had been exposed to continuous light for 24 h. Immunoblot analysis was performed as in (c). The figure was assembled from different blots (delineated by a black rectangle) and full-length blots are presented in Supplementary Fig. S8. (e) Real-time PCR analyses of 3-day-old etiolated WT (Col-0) and mutant seedlings that had been exposed to continuous light for 24 h. Real-time PCR was performed with primers specific for the nuclear genes LHCB1.2, LHCB2.1, LHCB6, LHCA5, PSBP-1 and PSBTn, and the plastid genes psaA and atpB. Note that the primers for LHCB2.1 also amplify LHCB2.2 mRNA. Expression values are reported relative to the corresponding transcript levels in the WT and were normalized with respect to the expression level of ACTIN2. Data are shown as mean values?±?SD from three different plant pools.

Reactant: Arabidopsis thaliana (Thale cress)
Application: Western Blotting
Pudmed ID: 33322466
Journal: Biomolecules
Figure Number: 7A
Published Date: 2020-12-11
First Author: Andreeva, A. A., Vankova, R., et al.
Impact Factor: None
Open PublicationImmunoblot analysis of the photosynthetic proteins on the basis of equal total Ponceau S dye stained blot with proteins from leaves of wild type plants and pap1 mutant grown on MS medium in Petri dishes for four weeks under a 16 h light/8 h dark photoperiod at 23 °C with 100 ?E m?2 s?1. Proteins were visualized by immunoblotting using antibodies specific for RbcL, PsaB, PsbD, AtpB, RpoB, AccD and Lhcb2.4 proteins.

Reactant: Arabidopsis thaliana (Thale cress)
Application: Western Blotting
Pudmed ID: 33629953
Journal: Elife
Figure Number: 6A
Published Date: 2021-02-25
First Author: Pipitone, R., Eicke, S., et al.
Impact Factor: 7.448
Open PublicationAccumulation dynamics of photosynthesis-related proteins during de-etiolation.Three-day-old etiolated seedlings of Arabidopsis thaliana were illuminated for 0 hr (T0), 4 hr (T4), 8 hr (T8), 12 hr (T12), 24 hr (T24), 48 hr (T48), 72 hr (T72), and 96 hr (T96) under white light (40 µmol/m2/s). (A) Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane and immunodetected with antibodies against PsbA, PsbD, PsbO, PetC, PsaD, PsaC, Lhcb2, AtpC, ELIP, POR, phyA, HY5, and ACTIN proteins. (B–C) Quantification of PsbA, PetC, and PsaC during de-etiolation. Heatmap (B) was generated after normalization of the amount of each protein relative to the last time point (T96). Graph (C) corresponds to the absolute quantification of proteins at T96. Error bars indicate ± SD (n = 3). Quantification of photosystem-related proteins during de-etiolation is detailed in Figure 6—figure supplement 1.Figure 6—source data 1.Quantitative data for immunoblot analysis.Quantitative data for immunoblot analysis.Quantification of photosynthesis-related proteins.(A) Immunodetection of PsbA, PetC, and PsaC during de-etiolation. Dilutions were used for the later time points to avoid saturation of the signal. (B) Different bands were detected by Amersham Imager program and quantified by Image QuantTL (Amersham). (C) Calibration curves were created using recombinant proteins (Agrisera). Calibration curve composition: PsbA 10 ng (A; lane a), 5 ng (b), 2.5 ng (c), and 1.25 ng (d); PetC 10 ng (e), 5 ng (f), 2.5 ng (g), and 1.25 ng (h); PsaC 3 ng (i), 1.5 ng (l), 0.75 ng (m), and 0.325 ng (n). Data indicate mean ± SD (n = 3–4). Raw data and calculations are shown in Figure 6—source data 1.
Additional information
There is a confirmed cross-reaction with TLA1 protein in Chlamydomonas reinhardtii.
For samples with a very low PSII content theremight be detection problems independent of the antibody. PSII proteins can vary in level depending upon liquid culture conditions. When the cells are in a stationary phase PSII content can drop to a very low level.
Background
D2 protein (PsbD) forms the reaction core of PSII (Photosystem II) as a heterodimer with the D1 protein (PsbA). PsbD is homologous to the D1 protein, with slightly higher molecular mass of about 39.5 kDa. Accumulation of D2 protein is an important step in the assemply of the PSII reaction centre complex.
Product citations
Bychkov et al. (2022) The role of PAP4/FSD3 and PAP9/FSD2 in heat stress responses of chloroplast genes. Plant Sci. 2022 Sep;322:111359. doi: 10.1016/j.plantsci.2022.111359. Epub 2022 Jun 20. PMID: 35738478.
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Chen, Liu & Liu (2021) Loss-Function of EGY1 Results in Photosynthesis Damage through Reducing Stability of Photosystem II in Arabidopsis thaliana. Russ J Plant Physiol (2021). https://doi.org/10.1134/S1021443721060029
Li et al. (2021). Physiological responses of Skeletonema costatum to the interactions of seawater acidification and the combination of photoperiod and temperature. Biogeosciences, 18, 1439–1449, 2021 https://doi.org/10.5194/bg-18-1439-2021
Pipitone et al. (2021). A multifaceted analysis reveals two distinct phases of chloroplast biogenesis during de-etiolation in Arabidopsis. Elife. 2021 Feb 25;10:e62709. doi: 10.7554/eLife.62709. PMID: 33629953; PMCID: PMC7906606.
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